Nicotinamide adenine dinucleotide phosphate (reduced form, NADPH) is a kind of coenzyme participating in reactions of a lot of oxidoreductase and dehydrogenase as an electron donor to provide reducing power together with NADH sharing a nicotinamide adenine dinucleotide structure. Oxides (NAD+ and NADP+) of these coenzymes perform an important function of receiving energy generated in biological catabolism in the form of electron and proton and participate in the reaction of oxidoreductase as an electron acceptor.
As well known, a main metabolism of a biological system is configured of a generation process of main precursors and energy by oxidation (catabolism) of nutrients inputted from the outside and a synthesis process (anabolism, which is mainly reduction reaction) of bio-molecules using the generated precursors and energy. A form of bio-energy generated in the oxidation (catabolism) process and consumed in the synthesis (anabolism) process is the electron and the proton, and the main carriers of these compounds are NADP(H) and NAD(H). The reason that two kinds of biological coenzymes capable of mediating chemically the same reaction exist is known as evolutional selection for appropriately distributing oxidizing (or reducing) power required for the catabolism and reducing (or oxidizing) power required for the anabolism and efficiently adjusting a flow of energy.
Therefore, it is known that since catabolism is mainly the oxidation reaction, and anabolism is mainly the reduction reaction, a molar ratio of NADPH is relatively higher than that of NADP+ in vivo and a molar ratio of NAD+ is relatively higher than that of NADH. In addition, in the metabolism/energy related processes in organisms growing through anaerobic respiration or fermentation and most of the organisms using an inorganic substance as an energy donor as well as in a respiration process in a general oxygen dependent cell using oxygen as a final electron acceptor, a coenzyme used as a high energy intermediate compound or an electron acceptor/donor is NADP(H) or NAD(H).
It is known that since it is very important to maintain a balance between these coenzymes due to specialized functions of the two kinds of coenzymes (NADP(H) or NAD(H)) in vivo, a salvage pathway (a pathway in which reduced NADPH reduces NAD+ or a reverse pathway thereof) capable of inducing an oxidized/reduced form of each other is present between the NADP(H) and NAD(H).
Therefore, a ratio of oxidized/reduced coenzyme is used as a very important biological activity marker in all of the cells in the ecosystem. That is, whether or not the cell maintains a normal biological activity and a difference in the biological activity may be prescribed by a method of quantifying a concentration of these coenzymes in vivo. As described above, it is known that in the case in which an accurate detection method for any one of the two kinds of coenzymes is present, concentrations of these coenzymes or a content of each of the oxidized/reduced form thereof may be quantified due to biochemical association between these coenzymes.
That is, as the function of these coenzymes in vivo, in the case of NAD(H), NAD(H) plays a very important role in energy generation or energy storage in a glycolytic process, which is a first process of the central metabolism in living organisms, a pentose phosphate pathway, and a TCA cycle. In the case of NADP(H), it is known that since NADP(H) is used to synthesize important biopolymers such as fatty acids, amino acids, and nucleic acids, the NADP(H) may be an important marker to activity measurement of various cells in a human body and as well as the physiological activity, presence or absence of metabolic disorders according to activity ratios thereof, analogy of a carcinogenesis process, a probe, or the like. In the case of living cells, since a relative difference in the concentration of these coenzymes in vivo or a ratio of oxidation/reduction is clear and is an absolute factor in the physiological activity, the coenzymes may also be a useful marker in determining a presence or absence of a cell in a specific sample (water quality analysis or confirmation of food contaminations) or a degree of the cell (possibility of causing a disease).
Therefore, a measuring method for accurate quantities of the two kinds of coenzymes may be widely used in various fields, such that various researches into the development of a measuring method of the coenzymes have been conducted, and various methods has been attempted in order to develop methods for detecting NADP(H) which has a relatively excellent optical property and of which a compound itself has a fluorescence intensity of a predetermined level. As described above, the accurate detection method of NADP(H) may provide an indirect detection method for oxidized NADP+, NADH or NAD+ by the coupling of salvage enzymes in addition to being used to measure NADPH itself.
A general method used for detection of NADPH, which is a main biological activity marker, is a method of measuring absorbance using a natural wavelength of NADPH itself or measuring fluorescence. The method of measuring absorbance is a method of measuring a light absorption degree at a wavelength of 340 to 345 nm, which is the intrinsic wavelength, using a UV spectrophotometer and then determining a quantity using a standard curve. In the case of the method for measuring the fluorescence, a method of irradiating light at about 350 nm at which maximum fluorescence appears through excitation scanning, and then measuring relative value at an emission wavelength (about 450 nm) to determining an amount corresponding thereto in a standard curve may be used.
The above-mentioned analysis methods have an advantage in that since addition of a separate substrate is not required, the measurement may be relatively simple, but have a disadvantage in that since the absorbance and fluorescence value of NADPH itself are low, a relatively accurate value may be measured in only pure reactant composition (including only a buffer and the coenzyme). Basically, since molecular extinction coefficient or quantum yield is low, the analysis method has a significant disadvantage in that a relatively large amount of samples of a predetermined level or more has been required.
The problem means that it is difficult to directly use the method in various biological or environmental samples unlike an artificial experimental sample in which an accurate composition ratio of the composition is known. The reason is that substance absorbing light or fluorescing at these wavelengths that are known as the intrinsic property of NADPH or various chemicals in environment may cause interference. Therefore, an S/N ratio (signal/noise ratio) may be frequently reduced, and sensitivity may be reduced, such that the measurement may be performed only in the case in which a concentration of NADPH in the sample is relatively high.
Another disadvantage of the current used method is that in the case in which a small amount of coenzyme exists, a measurement time may be delayed due to a complicated pre-treatment process generally used in order to solve a problem of NADPH having a low optical property. As described above, in the case in which an amount of coenzymes to be measured is small, the pre-treatment process for removing interference factors affecting the signal/noise ratio in vivo and in vitro is essential. This pre-treatment process includes steps such as a step of centrifugation, a step of inducing aggregation of the interference substances, or a step of concentration, and a time required to perform the pre-treatment process is 30 minutes to several hours (3 to 4 hours) according to the used process.
Through the process, interference may be partially reduced, and NADPH may be concentrated, but natural oxidation of NADPH according to the pre-treatment time may cause another problem. As well known, since NADPH having strong reducing power may easily provide the reducing power to other substances, NADPH has a chemical property in that it may be easily oxidized when it is exposed to air.
Therefore, there is a problem in that as the pre-treatment time become long, NADPH is converted into NADP+, which is relatively difficult to be measured, due to the natural oxidation by air, instability according to pH of the buffer, and reaction results with oxides in the reaction solution. Therefore, addition of a reagent suppressing oxidation of NADPH or inducing structural stability is separately required.
As a partial complementary measure of these problems, many kits currently sold in the market use the principle of coupling reaction in which cyclase for inducing reduction of the oxidized coenzyme (NADP+) or various dehydrogenases inducing reduction of NADP+ to NADPH through an oxidation reaction of a specific substrate using NADP+ as a coenzyme are included. However, there are disadvantages in that the enzyme source used in these reactions is expensive, stability is low, and another substrate should be added for measurement of the coenzyme.
Particularly, in the case of cyclase, ATP, which is another expensive coenzyme, should be necessarily added. As expected, during the process, since oxidized NADP+ already existing in the sample is also reduced and measured, a total amount of NADP+/NADPH is measured, instead of an absolute amount of NADPH in the sample or a relative ratio of the NADPH to the oxidized coenzyme.
In the kit sold on the market, as a complementary measure of low absorbance (fluorescence) property of NADPH, a method of inducing conversion of a specific fluorescent substrate using NADPH as a coenzyme and measuring fluorescence of a specific product increased accordingly may be used. In the above mentioned method, there is an advantage in that NADPH itself having low sensitivity that is difficult to be measured may be detected at a relatively high sensitivity using an artificial substrate having a high quantum yield, but there is a disadvantage in that enzyme to be coupled is required and an expensive artificial fluorescent substrate should be used, such that there are many limitations in using the method.